The goal of the proposed research is to determine whether technology can be developed to use an electrostatic phase-contrast aperture ("quarter-wave plate") for in-focus phase contrast in electron microscopy of biological macromolecules. The advantages of using in-focus phase contrast for applications in biomedical research are expected to be: Images of protein complexes as small as 250 kDa should be more easily identified and selected for computer processing, due to the improved contrast transfer function (CTF) at low spatial frequencies that is provided by in-focus phase contrast. Structural information at a resolution of 0.8 nm will no longer be corrupted or lost due to use of large value of defocus to achieve the needed amount of phase contrast. The improved image quality may result in improvement in the ability to detect structural compositional) and conformational differences amongst particles in a non-homogeneous sample. The final goal for the proposed research is to determine whether image-data collected with the help of a phase-contrast aperture can be used to obtain interpretable, three-dimensional reconstructions of multiprotein complexes as small as 250 kDa at a resolution of 0.8 nm. This work is based upon a novel, 2-electrode "drift tube" design that has some advantages in terms of fabrication at sub-micrometer dimensions. Based on our preliminary studies, we now propose to combine this device with an electron optical column that is modified in order to provide the desired match between the size of the electron diffraction pattern and the size of the microfabricated aperture. We will use the ratio of Fourier-transform amplitudes at a resolution "g" and at zero frequency [i.e. F(g)/F(0)] to quantitatively evaluate the effect that charging of the aperture has on contrast transfer at high resolution, and we will then use this quantitative assay to evaluate the effectiveness of alternative methods to avoid unwanted charging of the device. When we have demonstrated that the device can be used to produce a nearly flat CTF between ~1/(30 nm) and 1/(0.8 nm), we will automate all steps needed to use it for low-dose EM. The function of automation is to ensure that in-focus phase contrast is no more difficult to use than is defocus based phase contrast. In addition, we will test the performance of in-focus phase contrast on multiprotein complexes of various sizes in order to determine whether it is possible to obtain 3-D reconstructions at a resolution of 0.8 nm for particles as "small" as 250 kDa.